Abstract Previous studies have suggested that NK cells may limit T cell responses by their ability to eradicate dendritic cells, as demonstrated by NK cell-mediated killing of dendritic cells generated from mouse bone marrow cells or human monocytes with GM-CSF. In the present study, we demonstrated that conventional dendritic cells, generated in vitro with Flt3 ligand or from spleens, were resistant to NK cell-mediated lysis. However, upon stimulation with GM-CSF, NK cells could mediate lysis of these dendritic cells. GM-CSF-stimulated Flt3 ligand dendritic cells or splenic dendritic cells increased surface expression of costimulatory molecules and known NK cell ligands. Likewise, NK cells could target dendritic cells in vivo, which could be inhibited, in part, by anti-GM-CSF antibodies. The blocking of CD54 or CD226 inhibited NK cell-mediated cytotoxicity of the GM-CSF-stimulated Flt3 ligand conventional dendritic cells. Furthermore, the CD226+NKG2A− subset of NK cells was selectively better at targeting GM-CSF-stimulated Flt3 ligand conventional dendritic cells. However, CD155, a known ligand for CD226, could also act as an inhibitor of NK cell-mediated lysis, as dendritic cells lacking CD155 were more sensitive to NK cell-mediated lysis than wild-type dendritic cells. We hypothesize that by only permitting a subset of NK cells to target activated dendritic cells during inflammation, this would allow the immune system to balance between dendritic cells able to drive adaptive immune responses and dendritic cells targeted for elimination by NK cells to hinder, e.g., spread of infection.